True human antibody specific for interleukin 1 alpha

ABSTRACT

Fully human monoclonal Abs includes (i) an antigen-binding variable region that exhibits very high binding affinity for IL-1α and (ii) a constant region that is effective at both activating the complement system though C1q binding and binding to several different Fc receptors.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a by-pass continuation under 35 U.S.C. 111(a) ofinternational patent application number PCT/CA2021/051382 filed Oct. 4,2021, which claims priority to claims priority to Canadian PatentApplication number 3,095,679, filed Oct. 7, 2020, the entire contents ofwhich are hereby incorporated by reference.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

Not applicable.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in XML format and is hereby incorporated byreference in its entirety. Said XML copy, created on Mar. 24, 2023, isnamed 5407-0412_SL.xml and is 7,248 bytes in size.

FIELD OF THE INVENTION

The invention relates generally to the fields of immunology andantibodies (Abs).

BACKGROUND

Interleukin 1 alpha (IL-1α) is a pro-inflammatory cytokine that plays arole in a number of different activities including inflammation, immuneresponses, tumor metastasis, and hematopoiesis. IgG autoantibodiesagainst IL-1α occur naturally in the general human population and arethought to be beneficial in a number of different diseases which involvesterile inflammation.

SUMMARY

The amino acid sequence encoding the light and heavy chain variableregion of a monoclonal Ab (mAb) that binds human IL-1α with highaffinity was discovered. Accordingly, described herein are purifiedhuman mAbs including an antigen-binding variable region that (i)exhibits very high binding affinity for human IL-1α and (ii) comprises alight chain variable region including the amino acid sequence of SEQ IDNO:1 (or the CDRs thereof) and a heavy chain variable region includingthe amino acid sequence of SEQ ID NO:2 (or the CDRs thereof).

Also described herein is a set of isolated nucleic acids including afirst nucleic acid encoding the heavy chain of a human mAb thatspecifically binds to IL-1α, and a second nucleic acid encoding thelight chain of the human mAb that specifically binds to human IL-1α. Thefirst nucleic acid can encode the amino acid sequence of SEQ ID NO: 1(or the CDRs thereof) and the second nucleic acid can encode the aminoacid sequence of SEQ ID NO:2 (or the CDRs thereof).

In another aspect, described herein are expression vectors which includeboth a nucleic acid encoding the amino acid sequence of SEQ ID NO:1 (orthe CDRs thereof) and a nucleic acid encoding the amino acid sequence ofSEQ ID NO:2 (or the CDRs thereof). Also described herein are a set ofexpression vectors including a first expression vector encoding theamino acid sequence of SEQ ID NO:1 (or the CDRs thereof) and a secondexpression vector encoding the amino acid sequence of SEQ ID NO:2 (orthe CDRs thereof).

Additionally described herein is an isolated host cell (e.g. a mammaliancell such as a CHO cell) including a nucleic acid encoding the aminoacid sequence of SEQ ID NO:1 (or the CDRs thereof) and a nucleic acidencoding the amino acid sequence of SEQ ID NO:2 (or the CDRs thereof).

Unless otherwise defined, all technical terms used herein have the samemeaning as commonly understood by one of ordinary skill in the art towhich this invention belongs. Commonly understood definitions ofbiological terms can be found in Rieger et al., Glossary of Genetics:Classical and Molecular, 5th edition, Springer-Verlag: New York, 1991;and Lewin, Genes V, Oxford University Press: New York, 1994.

As used herein, the word “a” or “an” before a noun represents one ormore of the particular noun. For example, the phrase “an antibody”represents “one or more antibodies.”

By the term “antibody” or “Ab” is meant any immunoglobulin (e.g., human,rodent, cartilaginous fish, or camelid antibodies) or conjugate thereof,that specifically binds to an antigen (e.g., human IL-1α). A widevariety of Abs are known by those skilled in the art. Non-limitingexamples of Abs include: monoclonal Abs (e.g., including full-lengthAbs), polyclonal Abs, multi-specific Abs (e.g., bi-specific Abs),single-chain Abs (e.g., single-domain Abs, camelid Abs, andcartilaginous fish Abs), chimeric (e.g., humanized) Abs, and fully humanAbs including those that can be found or induced in human beings (i.e.,true human Abs). The term antibody also includes Ab conjugates (e.g., anAb conjugated to a stabilizing protein, a label, or a therapeutic agent(e.g., any of the therapeutic agents described herein or known in theart)).

By the term “antigen-binding fragment” is meant any portion of afull-length Ab that contains at least one variable domain [e.g., avariable domain of a mammalian (e.g., human, mouse, rat, rabbit, orgoat) heavy or light chain immunoglobulin, a camelid variableantigen-binding domain (VHH), or a cartilaginous fish immunoglobulin newantigen receptor (Ig-NAR) domain] that is capable of specificallybinding to an antigen. For example, an antigen-binding fragmentdescribed herein can include at least part of an Ab Fc region that issufficient to mediate antibody-dependent cell-mediated cytotoxicity(ADCC) and/or complement-dependent cytotoxicity (CDC) in a mammal (e.g.,a human) and/or is conjugated to a therapeutic agent (e.g., any of thetherapeutic agents described herein or known in the art). As anotherexample, an antigen-binding fragment described herein can include atleast part of an Ab Fc region that does not mediate ADCC and/or CDC in amammal (e.g., a human). Non-limiting examples of Ab fragments includeFab, Fab′, F(ab′)2, Fv fragments, diabodies, linear antibodies, andmulti-specific Ab formed from Ab fragments. Additional Ab fragmentscontaining at least one camelid VHH domain or at least one cartilaginousfish Ig-NAR domain include mini-bodies, micro-antibodies,subnano-antibodies, and nano-antibodies, and any of the other forms ofAbs described in U.S. Patent Application Publication No. 2010/0092470.

By the term “human antibody” is meant an Ab that is encoded by a nucleicacid (e.g., rearranged human immunoglobulin heavy or light chain locus)present in the genome of a human. In some embodiments, a human Ab isproduced in a mammalian (e.g., human) cell culture (e.g., a Chinesehamster ovary cell line). In some embodiments, a human Ab is produced ina non-human cell (e.g., a mouse or hamster cell line). In someembodiments, a human Ab is produced in a bacterial or yeast cell.

By the term “single-chain antibody” is meant a single polypeptide thatcontains at least one variable binding domain) that is capable ofspecifically binding to an antigen. Non-limiting examples ofsingle-chain Abs are described herein, and are known in the art (see,for example, the antibodies described in U.S. Patent Publication No.2010/0092470).

An Ab or antigen-binding fragment thereof “specifically binds” or “bindsspecifically” to a particular antigen, e.g., human IL-1α (via theepitope of a full-length antibody including the light and heavy chainvariable regions described herein binds), when it binds to that antigen,but recognizes and binds to a lesser extent (e.g., does not recognizeand bind) to other molecules in a sample. In some embodiments, an Ab oran antigen-binding fragment thereof selectively binds to an epitope withan affinity (KD) equal to or less than 1×10⁻¹⁰ M (e.g., less than1×10⁻¹¹ M or less than 1×10⁻¹² M) in phosphate buffered saline (e.g., asdetermined by surface plasmon resonance). The ability of an Ab orantigen-binding fragment to specifically bind a protein epitope may bedetermined using any of the methods known in the art or those methodsdescribed herein.

By the term “complementary determining region” or “CDR” is meant aregion within an Ig (heavy or light chain Ig) that forms part of anantigen-binding site in an Ab or antigen-binding fragment thereof. As isknown in the art, a heavy chain Ig contains three CDRs: CDR1, CDR2, andCDR3, respectively, and a light chain Ig contains three CDRs: CDR1,CDR2, and CDR3. In any Ab or antigen-binding fragment thereof, the threeCDRs from the heavy chain Ig and the three CDRs from the light chain Igtogether form an antigen-binding site in the Ab or antigen-bindingfragment thereof. The Kabat Database is one system used in the art tonumber CDR sequences present in a light chain Ig or a heavy chain Ig.

Although methods and materials similar or equivalent to those describedherein can be used in the practice or testing of the present invention,suitable methods and materials are described below. All applications andpublications mentioned herein are incorporated by reference in theirentirety. In the case of conflict, the present specification, includingdefinitions will control. In addition, the particular embodimentsdiscussed below are illustrative only and not intended to be limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments of the present disclosure will now be described, by way ofexample only, with reference to the attached Figures.

FIG. 1 is a graph showing the results of an Octet Red 96 assay indicatedthat the binding affinity (KD) for IL-1α of XIA13 was 6.25×10⁻¹¹.

FIG. 2 is a graph showing the results of an Octet Red 96 assay indicatedthat the binding affinity (KD) for the neonatal Fc receptor (FcRn) ofXIA13 was 2.0810⁻⁷.

DETAILED DESCRIPTION

Described herein are compositions and methods relating to fully (true)human mAbs that include an antigen-binding variable region that exhibitsvery high binding affinity for IL-1α. The below described preferredembodiments illustrate adaptation of these compositions and methods.Nonetheless, from the description of these embodiments, other aspects ofthe invention can be made and/or practiced based on the descriptionprovided below.

Methods involving conventional immunological and molecular biologicaltechniques are described herein. Immunological methods (for example,assays for detection and localization of antigen-Ab complexes,immunoprecipitation, immunoblotting, and the like) are generally knownin the art and described in methodology treatises such as CurrentProtocols in Immunology, Coligan et al., ed., John Wiley & Sons, NewYork. Techniques of molecular biology are described in detail intreatises such as Molecular Cloning: A Laboratory Manual, 2nd ed., vol.1-3, Sambrook et al., ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 2001; and Current Protocols in Molecular Biology,Ausubel et al., ed., Greene Publishing and Wiley-Interscience, New York.Ab methods are described in Handbook of Therapeutic Abs, Dubel, S., ed.,Wiley-VCH, 2007. Cell culture techniques are generally known in the artand are described in detail in methodology treatises such as Culture ofAnimal Cells: A Manual of Basic Technique, 4th edition, by R IanFreshney, Wiley-Liss, Hoboken, N.J., 2000; and General Techniques ofCell Culture, by Maureen A Harrison and Ian F Rae, Cambridge UniversityPress, Cambridge, UK, 1994. Methods of protein purification arediscussed in Guide to Protein Purification: Methods in Enzymology, Vol.182, Deutscher M P, ed., Academic Press, San Diego, Calif., 1990.

A fully human mAb includes an antigen-binding variable region that (i)exhibits very high binding affinity for human IL-1α and (ii) comprises alight chain variable region including the amino acid sequence of SEQ IDNO:1 (or the CDRs thereof) and a heavy chain variable region includingthe amino acid sequence of SEQ ID NO:2 (or the CDRs thereof). The lightand heavy chain variable regions (which together form the Fab) describedherein can be joined to an Fc or portion thereof using conventionalmolecular biology techniques to fuse the desired Fc portion to the Fabor antigen-binding fragment. In this way, full-length immunoglobulinssuch as human IgG1 (e.g., IgG1a or IgG1b), IgG2 (e.g., IgG2a or IgG2b),IgG3 (e.g., IgG3a or IgG3b), IgG4 (e.g., IgG4a or IgG4b), IgD, IgA(e.g., IgA1, and IgA2), IgE, or IgM (e.g., dimeric, pentameric, andhexameric) (and the different allotypes of the foregoing) incorporatingthe light and heavy chain variable regions described herein can be made.

The mAb described herein might be affinity matured to enhance orotherwise alter their binding specificity by known methods such as VHand VL domain shuffling (Marks et al. Bio/Technology 10:779-783, 1992),random mutagenesis of the hypervariable regions (HVRs) and/or frameworkresidues (Barbas et al. Proc Nat. Acad. Sci. USA 91:3809-3813, 1994;Schier et al. Gene 169:147-155, 1995; Yelton et al. J. Immunol.155:1994-2004, 1995; Jackson et al., J. Immunol. 154(7):3310-9, 1995;and Hawkins et al, J. Mol. Biol. 226:889-896, 1992. Amino acid sequencevariants of an Ab may be prepared by introducing appropriate changesinto the nucleotide sequence encoding the Ab. In addition, modificationsto nucleic acid sequences encoding mAbs might be altered (e.g., withoutchanging the amino acid sequence of the mAb) for enhancing production ofthe mAb in certain expression systems (e.g., intron elimination and/orcodon optimization for a given expression system). The mAbs describedherein can also be modified by conjugation to another protein (e.g.,another mAb) or non-protein molecule. For example, a mAb might beconjugated to a water-soluble polymer such as polyethylene glycol or acarbon nanotube (See, e.g., Kam et al., Proc. Natl. Acad. Sci. USA 102:11600-11605, 2005). See, U.S. patent application Ser. No. 11/754,899.

Amino acid mutations may be introduced into the constant region of theseIgG subclasses. Amino acid mutations that can be introduced may be, forexample, those that enhance binding to FcK receptors (as described in,e.g., Proc. Natl. Acad. Sci. U.S.A. 103(11):4005-4010, 2006; MAbs 1(6):572-579, 2009; US 2010/0196362; US 2013/0108623; US 2014/0171623; US2014/0093496; and US 2014/0093959), or enhance or decrease binding toFcRn (as described in, e.g., J. Biol. Chem. 276(9):6591-6604, 2001; IntImmunol. 18(12):1759-1769, 2006; and J. Biol. Chem. 281(33):23514-23524,2006).

Two types of H chains are heterologously associated to produce abispecific Ab. The knobs-into-holes technology (as described in, e.g.,J. Immunol. Methods 248(1-2):7-15, 2001; and J. Biol. Chem. 285(27):20850-20859, 2010), the electrostatic repulsion technology (as describedin, e.g., WO 06/106905), the SEEDbody technology (as described in, e.g.,Protein Eng. Des. Sel. 23(4):195-202, 2010), and such may be used forheterologous association of two types of H chains via a CH3 domain. Anyof the Abs described herein may be those with a modified or deficientsugar chain. Examples of Abs having modified sugar chains includeglycosylation-engineered antibodies (as described in, e.g., WO99/54342), Abs with defucosylated sugar chains (as described in, e.g.,WO 00/61739, WO 02/31140, WO 06/067847, and WO 06/067913), and Abshaving a sugar chain with bisecting GlcNAc (as described in, e.g., WO02/79255). Known examples of methods for producing sugar chain-deficientIgG antibodies include the method of introducing a mutation toasparagine at EU numbering position 297 in the heavy chain (J. Clin.Pharmacol. 50(5): 494-506, 2010), and the method of producing IgG usingE. coli (J. Immunol. Methods 263(1-2):133-147, 2002; and J. Biol. Chem.285(27):20850-20859, 2010). Furthermore, heterogeneity accompanyingdeletion of C-terminal lysine in IgG, and heterogeneity accompanyingmispairing of disulfide bonds in the hinge region of IgG2 can bedecreased by introducing amino acid deletions/substitutions (asdescribed in, e.g., WO 09/041613). Any of the Abs or antigen-bindingfragments described herein includes at least one (e.g., one, two, three,four, five, or six) amino acids (e.g., an added, inserted, orsubstituted amino acid, e.g., not within a CDR) that are not present ina corresponding human Ab. Any of the Abs or antigen-binding fragmentsdescribed herein can also have at least one amino acid deleted (e.g., ascompared to a corresponding human Ab), e.g., a deletion from the N- orC-terminus of a light or heavy chain, or a deletion of an amino acidfrom a constant domain (e.g., Fc domain).

Preferably, to ensure that high titers of the human IL-1α-specific mAbcan be administered to a subject with minimal adverse effects, the mAbcompositions of the invention are at least 0.5, 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90,95, 96, 97, 98, 99, 99.9 or more percent by weight pure (excluding anyexcipients). The mAb compositions of the invention might include only asingle type of mAb (i.e., one produced from a single clonal B lymphocyteline). In addition to the human IL-1α mAb, the Ab compositions of theinvention might also include other mAbs that specifically bind antigensother than human IL-1α.

To modify or enhance its function, the mAb might be conjugated withanother molecule such as a cytotoxin or detectable label. A humanIL-1α-specific mAb might be conjugated with one or more cytotoxins tomore effectively kill cells expressing IL-1α. Cytotoxins for use in theinvention can be any cytotoxic agent (e.g., molecule that can kill acell after contacting the cell) that can be conjugated to a human IL-1αspecific mAb. Examples of cytotoxins include, without limitation,radionuclides (e.g., ³⁵S, ¹⁴C, ³²P, ¹²⁵I, ¹³¹I, ⁹⁰Y, ⁸⁹Zr, ²⁰¹Tl, ¹⁸⁶Re,¹⁸⁸Re, ⁵⁷Cu, ²¹³Bi, and ²¹¹At), conjugated radionuclides, andchemotherapeutic agents. Further examples of cytotoxins include, but arenot limited to, antimetabolites (e.g., 5-fluorouricil (5-FU),methotrexate (MTX), fludarabine, etc.), anti-microtubule agents (e.g.,vincristine, vinblastine, colchicine, taxanes (such as paclitaxel anddocetaxel), etc.), alkylating agents (e.g., cyclophosphamide, melphalan,bischloroethylnitrosurea (BCNU), etc.), platinum agents (e.g., cisplatin(also termed cDDP), carboplatin, oxaliplatin, JM-216, CI-973, etc.),anthracyclines (e.g., doxorubicin, daunorubicin, etc.), antibioticagents (e.g., mitomycin-C), topoisomerase inhibitors (e.g., etoposide,teniposide, and camptothecins), or other cytotoxic agents such as ricin,diphtheria toxin (DT), Pseudomonas exotoxin (PE) A, PE40, abrin,saporin, pokeweed viral protein, ethidium bromide, glucocorticoid,anthrax toxin and others. See, e.g., U.S. Pat. No. 5,932,188.

The human IL-1α specific mAb can also be conjugated to a detectablelabel. Useful detectable labels in the present invention include biotinor streptavidin, magnetic beads, fluorescent dyes (e.g., fluoresceinisothiocyanate, Texas red, rhodamine, green fluorescent protein, and thelike), radiolabels (e.g., ³H, ¹²⁵I, ³⁵, ¹⁴C, ³²P, ¹¹¹In, ⁹⁷Ru, ⁶⁷Ga,⁶⁸Ga, or ⁷²As), radiopaque substances such as metals for radioimaging,paramagnetic agents for magnetic resonance imaging, enzymes (e.g.,horseradish peroxidase, alkaline phosphatase and others commonly used inan ELISA), and colorimetric labels such as colloidal gold or coloredglass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads.Means of detecting such labels are well known to those of skill in theart. Thus, for example, radiolabels may be detected using photographicfilm or scintillation counters. Fluorescent markers may also be used andcan be detected using a photodetector to detect emitted illumination.Enzymatic labels are typically detected by providing the enzyme with asubstrate and detecting the reaction product produced by the action ofthe enzyme on the substrate, and colorimetric labels are detected bysimply visualizing the colored label.

The present invention also encompasses nucleic acid molecules encodingthe mAb specific for human IL-1α. Although the same nucleic acidmolecule might encode both the heavy and light chains of a humanIL-1α-specific mAb, a set of two different nucleic acid molecules, oneencoding the heavy chain and the other encoding the light chain mightalso be used. Any other suitable nucleic acid that encodes the aminoacid sequences of the mAb described herein might also be used.

For production of mAbs, the nucleic acid molecules encoding the heavyand light chains might be incorporated into an expression vector in anorientation wherein such nucleic acid molecules are operatively linkedto expression control sequences such as transcriptional andtranslational control sequences. Examples of expression vectors includevectors derived from plasmids and vectors derived from viruses such asadenoviruses, adeno-associated viruses, and retroviruses. The nucleicacid molecules encoding a light chain and a heavy chain might beincorporated into a single vector or different vectors. The vectors ofthe invention might also include regulatory sequences such as promotersand/or enhancers (see, U.S. Pat. Nos. 5,168,062, 4,510,245 and4,968,615), selectable markers, or sequences encoding affinity tags (forfacilitating purification) or a detectable label.

For production of mAbs, the vectors of the invention can be introducedinto a suitable host cell, e.g., a prokaryotic cell such as a bacteriaor, preferably, a eukaryotic cell such as mammalian, plant, or yeasthost cell. Examples of methods for introducing heterologouspolynucleotides into host cells include use of viral vectors,electroporation, encapsulation of the polynucleotide(s) in liposomes,dextran-mediated transfection, calcium phosphate precipitation,polybrene-mediated transfection, protoplast fusion,Agrobacterium-mediated transformation, biolistic transformation, anddirect microinjection of the DNA into nuclei. Mammalian cell lines arepresently preferred for expression of mAbs from vectors. Examples ofmammalian host cells include Chinese hamster ovary (CHO) cells (e.g.,the DG44 CHO cell line or the CHO-K1 cell line), HeLa cells, babyhamster kidney (BHK) cells, African green monkey kidney cells (COS),human hepatocellular carcinoma cells (e.g., Hep G2), NS0 cells, SP2cells, HEK-293T cells, 293 Freestyle cells, and NIH-3T3 cells. The mAbmight also be expressed in transgenic animals or plants. See, e.g., U.S.Pat. Nos. 5,827,690; 5,756,687; 5,750,172; 5,741,957; 6,046,037; and5,959,177.

The Abs and antigen-binding fragments described herein can be formulatedas pharmaceutical compositions which contain the Abs and antigen-bindingfragment and at least one pharmaceutically acceptable carrier (e.g., anon-natural pharmaceutically acceptable carrier). Non-limiting examplesof pharmaceutically acceptable carriers include sterilized water,physiological saline, stabilizers, excipients, antioxidants (e.g.,ascorbic acid), buffers (e.g., phosphate, citrate, histidine, and otherorganic acids), antiseptics, surfactants (e.g., PEG and Tween),chelating agents (e.g., EDTA or EGTA), and binders. Additional examplesof pharmaceutically acceptable carriers also includelow-molecular-weight polypeptides, proteins (e.g., serum albumin andgelatin), amino acids (e.g., glycine, glutamine, asparagine, glutamicacid, aspartic acid, methionine, arginine, and lysine), sugars andcarbohydrates (e.g., polysaccharides and monosaccharides), and sugaralcohols (e.g., mannitol and sorbitol). When preparing an aqueoussolution for injection, physiological saline and isotonic solutionscomprising glucose and other adjuvants such as D-sorbitol, D-mannose,D-mannitol, and sodium chloride may be used, and if necessary, incombination with appropriate solubilizers, such as alcohol (e.g.,ethanol), polyalcohols (e.g., propylene glycol and PEG), and nonionicsurfactants (e.g., polysorbate 80, polysorbate 20, poloxamer 188, andHCO-50). By mixing hyaluronidase into the formulation, a larger fluidvolume can be administered subcutaneously (see, e.g., Expert. Opin.Drug. Deliv. 4(4): 427-440, 2007).

The Abs and antigen-binding fragments provided herein may, e.g., beencapsulated in microcapsules (e.g., those made ofhydroxymethylcellulose, gelatin, and poly(methylmetacrylate)), orincorporated as components of colloidal drug delivery systems (e.g.,liposomes, albumin microspheres, microemulsion, nanoparticles, andnanocapsules) (see, for example, “Remington's Pharmaceutical Science16th edition”, Oslo Ed. (1980)). Methods for preparing thepharmaceutical compositions as controlled-release pharmaceutical agentsare also well-known, and such methods may be applied to the Abs andantigen-binding fragments of the present invention (see, e.g., Langer etal., J. Biomed. Mater. Res. 15: 267-277, 1981; Langer, Chemtech. 12:98-105, 1982; U.S. Pat. No. 3,773,919; European Patent ApplicationPublication No. EP 58,481; Sidman et al., Biopolymers 22: 547-556, 1983;and EP 133,988).

The pharmaceutical compositions provided herein can be formulated forintravenous, intraarterial, intradermally, subcutaneous, intramuscular,intraperitoneal, or oral administration.

EXAMPLES Example 1—Discovery of the Heavy and Light Chain VariableRegion Sequences of an Anti-Human IL-1α Ab

Plasma and peripheral blood mononuclear cells (PBMC) were isolated froma healthy human donor. The presence of anti-IL-1α antibodies in theplasma was confirmed by bead-based flow cytometric analysis usingstreptavidin magnetic beads that were conjugated with biotinylatedrecombinant human IL-1α. PBMCs were isolated from the donor's bloodusing Histopaque™ 1077 and Accuspin™ tubes, and cells were isolated froma portion of the PBMCs. RNA was extracted from the PBMCs as well as theB cells using conventional methods, and cDNA was prepared from the RNA.PCRs were performed on the cDNA using the procedure and primersdescribed in U.S. Pat. No. 9,453,217. The reverse primer used wasselected to specifically amplify IgG4 and IgG1 as the reactivities inthe plasma were isotyped to be predominantly of IgG4 subclass, with somesignal from IgG1 subclass. Both kappa and lambda light chain librarieswere made. Phage libraries were generated from IgG4-kappa overlaps andthree rounds of panning were performed without any phage amplificationin between the rounds. The input library diversity was found to be0.65e12. After round 2, half of the clones were counted on a plate, andthe remainder were subjected to an additional round of panning. Afterthree rounds of panning, 38 clones remained. An ELISA-based screeningwas performed with the phage supernatants on ELISA plates coated withrecombinant human IL-1α, with anti-FLAG antibody for detection of boundphages, and positive clones were identified. One of these-designatedXIA13—was selected and sequenced. The amino acid sequence of thevariable regions of the light and heavy chains of the human monoclonalantibody designated XIA13 is as follows with the CDRs (determined byIMGT/DomainGapAlign; Ehrenmann, F., Lefranc, M.-P. Cold Spring HarbProtoc., 2011(6):737-749 (2011). DOI:10.1101/pdb.prot5636.PMID:21632775) shown in bold and underlining:

>XIA13_Light Chain (SEQ ID NO: 1) DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNY LAWYQQKPGQP PKLLIY WA STRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC QQYY STPSTFGQGTKVEIKR >XIA13_Heavy Chain (SEQ ID NO: 2) QVQLVQSGAEVKKPGSSVKVSCKASGGRFTNYA ILWVRQAPGQGLQWLG G IIPIFD ETDHAQDFQDRLTITVDESMTTAYMELSSLRPEDTAIYYC AT GSNSYYGLY WGQGTLVTVSS

The XIA13 light chain thus has a CDR1 having the amino acid sequenceQSVLYSSNNKNY [SEQ ID NO:3], a CDR2 having the amino acid sequence WAS [,and a CDR3 having the amino acid sequence QQYYSTPST [SEQ ID NO:5].Similarly, the XIA13 heavy chain thus has a CDR1 having the amino acidsequence GGRFTNYA [SEQ ID NO:6], a CDR2 having the amino acid sequenceIIPIFDET [SEQ ID NO:7], and a CDR3 having the amino acid sequenceATGSNSYYGLY [SEQ ID NO:8].

Example 2—Characterization of XIA13

The results of an Octet Red 96 assay indicated that the binding affinity(KD) for IL-1α of XIA13 was 6.25×10⁻¹¹ (see FIG. 1 ). The results of anOctet Red 96 assay indicated that the binding affinity (KD) for theneonatal Fc receptor (FcRn) of XIA13 was 2.08×10⁻⁷ (see FIG. 2 ).

A HUVEC-based potency assay indicated that XIA13 IC₅₀ was 3.7 ng/ml.Briefly, 0.2×10⁶ HUVEC cells (Corning™ 354151)/ml were seeded in 96 wellflat bottom plate. XIA13 molecule was diluted to concentrations rangingfrom 610 pg/ml to 100 μg/ml. Diluted XIA13 was dosed to HUVEC cells inthe 96 well plate at final concentration ranges from 61 pg/ml to 10μg/ml. 0.5 ng/ml hIL-1α was applied to each well in the 96 well plateand the assay plate was incubated at 37° C./5% CO₂ for 18 hrs. HUVECcells were stained with anti-ICAM-1 antibody (eBioscience 12-0549, cloneHA58) and expression level of ICAM-1 was determined using flowcytometry. Data analysis was performed with FlowJo and IC 50 wascalculated using KaleidaGraph.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

What is claimed is:
 1. A pharmaceutical composition comprising apurified human monoclonal antibody that specifically binds tointerleukin 1 alpha (IL-1α), and comprises a light chain variable regionamino acid sequence comprising a CDR having the amino acid sequence ofSEQ ID NO:3, a CDR having the amino acid sequence WAS, and a CDR havingthe amino acid sequence of SEQ ID NO:5, and a heavy chain variableregion amino acid sequence comprising a CDR having the amino acidsequence of SEQ ID NO:6, a CDR having the amino acid sequence of SEQ IDNO:7, and a CDR having the amino acid sequence of SEQ ID NO:8.
 2. Thepharmaceutical composition of claim 1, wherein the light chain variableregion has the amino acid sequence of SEQ ID NO:1, and the heavy chainvariable region has the amino acid sequence of SEQ ID NO:2.